Emulsions as contrast agents and method of use

ABSTRACT

This invention relates to an oil-in-water emulsion that is of a water-insoluble gas-forming chemical and a stabilizer. The emulsion being capable of forming microbubbles of gas upon application of ultrasonic energy. This composition allows for site specific imaging as the image enhancing microbubbles can be released upon the application of ultrasonic energy at the specific location where the image is desired.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. Ser. No.08/072,535 filed on Jun. 4, 1993, now U.S. Pat. No. 5,716,597, thespecification of which is incorporated herein in its entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to diagnostic ultrasonic imaging and contrastagents for use thereof. More particularly, it relates to ultrasoniccontrast agents comprising emulsions capable of forming gas microbubblesupon the application of ultrasonic energy and methods for their use indiagnostic imaging.

2. Brief Description of the Background Art

Diagnostic ultrasonic imaging is based on the principal that waves ofsound energy can be focused upon an area of interest and reflected insuch a way as to produce an image thereof. The ultrasonic scannerutilized is placed on a body surface overlying the area to be imaged,and sound waves are directed toward that area. The scanner detectsreflected sound waves and translates the data into images. Whenultrasonic energy is transmitted through a substance, the amount ofenergy reflected depends upon the velocity of the transmission and theacoustic properties of the substance. Changes in the substance'sacoustic properties (e.g. variations in acoustic impedance) are mostprominent at the interfaces of different substances, such asliquid-solid or liquid-gas. Consequently, when ultrasonic energy isdirected through media, changes in acoustic properties will result inmore intense sound reflection signals for detection by the ultrasonicscanner.

Ultrasonic imaging agents of particular importance are composed ofgas-containing substances which, when injected into the circulatorysystem, provide improved sound reflection and image clarity. One classof gas-containing imaging agents consists of microspheres of gassurrounded by a shell made of a biocompatible substance. These are besttypified by ALBUNEX® (Molecular Biosystems, San Diego, Calif.: U.S. Pat.Nos. 4,572,203; 4,718,433; 4,744,958; 4,844,882 and 4,957,656) whichconsists of microspheres of air surrounded by albumin shells. Anothersuch microspheric imaging agent is described by Holmes, et al. Thesemicrospheres consist of either non-proteinaceous crosslinked orpolymerized amphipathic moieties forming micelles (PCT WO 92/17212) orcrosslinked proteins (PCT WO 92/17213), both of which encapsulate gassessuch as nitrogen, SF₆ and CF₄.

Another class of ultrasonic imaging agents can be described asmicroparticles of a solid or semi-solid substance containing gas whichis entrapped in the microparticle matrix during production. Glajich, etal. (U.S. Pat. No. 5,147,631) describe the formation of porous particlesof an inorganic material containing entrapped gas or liquid such as O₂,CF₄, perfluoroethane and argon. Erbel, et al. (U.S. Pat. No. 5,137,928)describe polyamino-dicarboxylic acid-co-imide derivatives capable ofentrapping gasses such as air, argon and krypton. Albayrak, et al.(European Patent Specification 0 357 163) describe crystalline complexesentrapping gasses such as nitrogen, krypton, SF₆, cyclopropane andpentane which are dissolved in an aqueous vehicle such as protein orglycerol causing the release of gas bubbles. The aqueous vehicle, nowcontaining a plurality of microbubbles of gas in solution, is then readyfor use as an injectable ultrasonic imaging agent. Stein, et al.(European Patent Specification 327 490) describe microparticlescontaining amyloses or synthetic biodegradable polymers entrappinggasses or liquids with a boiling point less than 60° C.

Another class of gas-containing imaging agents are lipid vesicles orliposomes. Unger (U.S. Pat. Nos. 5,088,499 and 5,123,414) describes theencapsulation of gasses or gaseous precursors in liposomes, moreparticularly liposomes which contain ionophores for activation ofgaseous precursors by way of a pH gradient. Henderson, et al. (PCT WO92/15824) describe lipid vesicles with gas-filled center cores.

Still another class of imaging agents is composed of microbubbles of gasin solution. For example, Tickner, et al. (U.S. Pat. No. 4,276,885)describe microbubbles dispersed in liquified gelatin. More-recently,Quay (PCT WO 93/05819) describes ultrasound imaging agents comprisingmicrobubbles of selected gasses in solution. In a specific embodiment,Quay describes the formation of a gas-liquid emulsion ofdecafluorobutane. Also disclosed therein are imaging agents comprisingaqueous dispersion of biocompatible gasses, some of which are gaseous atambient temperature and others of which become gaseous at the bodytemperature of the subject being imaged.

The efficiency of gas as an ultrasound imaging agent is described by J.Ophir and K. J. Parker, Contrast Agents in Diagnostic Ultrasound,Ultrasound in Medicine and Biology (1989), Vol. 15(4) p. 319-333.However, the disadvantages of using gas as an ultrasound imaging agenthave been and continue to be lacking of sufficient persistence of thegas in-vivo and in-vitro, and toxicity due to the introduction of gasinto the venous system.

The present invention relates to site specific oil-in-water emulsionsand is based on the unexpected observation that emulsions of gas-formingchemicals can be stabilized in the liquid state and will producemicrobubbles when subjected to ultrasonic energy. The advantages arethat such emulsions are more stable than most of the gas-containingimaging agents heretofor described, and their ability to formmicrobubbles when subjected to ultrasonic energy makes themsite-specific and inherently less toxic due to less overall gas beingintroduced into the venous system.

SUMMARY OF THE INVENTION

This invention provides an emulsion which can be used as an ultrasonicimaging agent. The emulsion is made of at least one water-insoluble gasforming chemical and at least one stabilizer. This emulsion is capableof forming microbubbles of gas upon application of ultrasonic energy.The stabilizer is either a hydrophobic or amphipathic compound having aboiling point higher than that of the gas-forming chemical and, whenpresent in the emulsion with the gas-forming chemical, acts as astabilizer (maintains the gas-forming chemical in the liquid state)until the application of ultrasonic energy. The stabilizer causes theeffective boiling point of the gas-forming chemical to be raised therebypreventing the volatilization of the gas-forming chemical until itreaches a temperature above its boiling point at atmospheric pressure(760 mm). In this way, upon application of ultrasonic energy, theemulsified chemical becomes volatilized and produces gas microbubbles.In a specific embodiment the water-insoluble gas forming chemical isperfluoropentane and the stabilizer is lecithin. This invention alsoprovides additional means to stabilize the emulsion for delivery to apatient. These means include delivery vehicles such as a natural polymermatrix, a synthetic polymer matrix or a liposome. More specifically, itis provided that the natural polymer matrix is an albumin matrix. Thisalbumin matrix can be derivatized to contain polyethylene glycol.

This invention also provides a method to enhance the contrast of tissuesand organs in an ultrasonic image comprising: (a) injecting at least onestabilized water insoluble gas forming chemical into a patient (b)applying a sufficient amount of ultrasonic energy to volatilize saidchemicals to release microbubbles; and (c) detecting an ultrasonicimage. The water insoluble gas forming chemical is stabilized with ahydrophobic or amphipathic stabilizer.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows the effects of increasing ultrasonic energy transmit poweron reflectivity of the ultrasonic signal (expressed as videobrightness)in the presence of an ALBUNEX® (Molecular Biosystems, SanDiego, Calif.) sample.

FIG. 1B shows the effects as described in FIG. 1A in the presence of theperfluoropentane emulsion of Example 1.

FIG. 2 shows the difference in video brightness observed when theemulsion of Example 5 is either continually exposed to ultrasonicenergy, or exposed only during 30 second intervals every 5 minutes.

FIG. 3 shows the difference in video brightness observed when Emulsion Cof Example 7 is either continually exposed to ultrasonic energy, orexposed only during 30 second intervals every 5 minutes.

FIG. 4 shows the ¹ H NMR spectrum of a CDCl₃ solution ofnonafluoro-t-butylmethane (C₄ F₉ CH₃).

FIG. 5 shows the ¹⁹ F NMR spectrum of a CDCl₃ solution ofnonafluoro-t-butylmethane (C₄ F₉ CH₃).

DETAILED DESCRIPTION OF THE INVENTION

We have now found that particularly effective site-specific ultrasoniccontrast agents may be obtained by preparing emulsions ofwater-insoluble gas-forming chemicals. These gas-forming chemicals arestabilized by emulsification with a stabilizer. Additionally, theemulsification of the gas-forming chemicals, which are for the most partinsoluble in water, serves to make the contrast agent more soluble andthus administrable to a patient. The water-insoluble gas-formingchemical must be capable of forming gas at the body temperature of theanimal being imaged and will generally have a boiling point below bodytemperature. As discussed herein, boiling point will refer to thetemperature at which the thermal energy of the molecules of a chemicalare great enough to overcome the cohesive forces that hold them togetherin a liquid state (or solid state for chemicals which sublime and thushave no liquid state) at atmospheric pressure (760 nm). A stabilizerhaving a boiling point higher than that of the gas-forming chemical isnecessary to stabilize the gas-forming chemical in the liquid stateuntil the application of ultrasonic energy. The stabilizer causes thetemperature at which the gas-forming chemical volatilizes to a gas to beraised to a temperature above its boiling point. In this way, thegas-forming chemical is actually both stabilized (maintained in a liquidstate above its boiling point) and destabilized (capable of beingvolatilized. upon exposure to ultrasonic energy) simultaneously. Whenthe emulsion of the present invention is volatilized by exposure toultrasonic energy, such as 50% transmit power at 5.0 MHz, gasmicrobubbles are formed and released from the emulsion therebyincreasing the ultrasonic reflectivity in the area being imaged.

The water-insoluble gas-forming chemicals useful in the presentinvention can be further characterized as being non-toxic,physiologically compatible and generally having a boiling point below37° C., and preferably between 26° C. and 34° C. Some of the gas-formingchemicals which would be useful in the present invention and theirboiling points at atmospheric pressure are:

                  TABLE 1                                                         ______________________________________                                        Gas-forming Chemical                                                                             Boiling Point, ° C.                                 ______________________________________                                        pentane            36                                                           1-pentene                                 30                                  perfluoropentane                         29.5                                 2-methyl butane (isopentane)              27.8                                tetramethylsilane                         26                                  2-bromo-1,1,1-trifluoroethane           26                                    dibromodifluoromethane                    25                                  fluorotrichloromethane                    24                                  2 H-perfluoro-t-butane                   13                                   cyclobutane                               12                                  heptafluoropropylbromide                  12                                  1-chloro-1,1,2,2-tetrafluoroethane     10.2                                   neopentane                                9.5                                 teflurane                                8                                    2-chloro-1,1,1-trifluoroethane          6.9                                   decafluorobutane                          4                                   butane                                   -.5                                  2-chloro-1,1,1,2-tetrafluoroethane     -12                                    2 H-heptafluoropropane                    -15                                 iodotrifluoromethane                     -22.5                                cyclopropane                             -33                                  perfluoroethylamine                       -35                                 octafluoropropane                        -36                                  SF.sub.6 (sulfur hexafluoride)                 -64                          ______________________________________                                    

The stabilizer of the present invention may be a hydrophobic oramphipathic (containing both hydrophobic and hydrophilic entities)compound. Hydrophobic compounds include di- and triglycerides; saturatedand unsaturated hydrocarbons; perfluorocarbons such as perfluorohexaneor perfluorodecalin; fats and fatty oils such as triolein.

Amphipathic compounds include phospholipids such as, phosphatidic acid,phosphatidylglycerol, and phosphatidylinositol; alkali salts of fattyacids; ionic surfactants such as sodium dodecyl sulfate; non-ionicsurfactants such as PLURONIC® F-68 (trade name for poloxamer 188, ablock copolymer of polyoxyethylene and polyoxypropylene ##STR1## whereinthe average value of n=75 and the average value of b=30 such that theaverage molecular weight of said compound is 8350) and polysorbate 80;zwitterionic surfactants such as phosphatidylcholine (lecithin),phospatidylethanolamine and phosphatidylserine; amino acid polymers orproteins with hydrophilic and hydrophobic moieties such as albumin.

Amphipathic compounds which are particularly useful as stabilizers offluorinated gas-forming compounds are themselves fluorinated. Thesecompounds act as both stabilizers and solubilizers of fluorinatedgas-forming compounds, due to the fluorine-fluorine interactions betweenthe two compounds. Such fluorinated stabilizers generally have ahydrophobic fluorocarbon chain connected to a hydrophilic moiety, suchas a polyether, sugar, carboxylate, sulfonate or a quaternary ammoniumgroup. Examples of fluorinated stabilizers can be found in U.S. Pat.Nos. 5,077,036, 5,080,855 and 4,987,154, each of which is incorporatedherein by reference.

When the boiling point of the gas-forming chemical is below thetemperature at which the emulsion is prepared and stored, such as lessthan 24° C., it is still possible to form a liquid-liquid oil-in-wateremulsion of the present invention by using a stabilizer which is capableof strong hydrophobic interations with the gas-forming chemical whichwill maintain the gas-forming chemical in a liquid state above itsboiling point. Particularly useful stabilizers for this purpose are C5to C20 perfluorocarbons or hydrocarbons and can be either hydrophobic oramphipathic.

The stabilizer may be used singly or in various combinations in theemulsions of the present invention. However, when the stabilizer is ahydrophobic compound, it will be necessary to also have present asurface active agent either within the emulsion or in association withthe emulsion in order for the emulsion to be soluble and thusphysiologically tolerated. Surface active agents, or surfactants, arecharacterized as being substances that lower the surface tension betweentwo liquids. A surface active agent will generally be an amphipathiccompound as described above or may also be a cationic or anioniccompound. Additionally, a surfactant and a co-surfactant combination,such as phosphatidylcholine and PLURONIC® F-68 is also contemplated.

When the stabilizer is amphipathic, the presence of an additionalhydrophobic compound is generally not necessary. In particular, thechemical PLURONIC® F-68 has been found to sufficiently solubilize andstabilize the gas-forming chemical in the absence of an additionalhydrophobic compound.

The amount of stabilizer present in the emulsion of the presentinvention will vary over a wide range of concentrations depending on theconcentration and properties of the other components of the emulsion andwill be principally dependent on the amount and characteristics of thegas-forming chemical. This is exemplified in the example section.

Optionally present in the emulsion are viscosifiers which are generallypolyalcohols or carbohydrates such as glycerol, sorbitol, lactose,sucrose and dextrans, and preferably glycerol at a concentration between5-15% (w/v). Other optional constituents are anti-oxidants such asα-tocopherol, preferably at a concentration of 0.1 to 0.25% (w/v). Stillanother class of optional components are compounds which impart organ ortissue target specificity to the emulsion. These compounds may includesteroids such as cholesterol, proteins, lipoproteins and antibodies.

The emulsion of the present invention may be useful as an ultrasonicimaging agent either by itself or in combination with a delivery vehiclewhich may be used to impart greater stability, both in-vivo andin-vitro, or tissue or organ target specificity. One such deliveryvehicle can be made from a natural polymer which forms a matrix, such asan albumin matrix, with multiple chambers which contain the emulsion ofa gas-forming chemical. The surface of the albumin matrix so describedmay also be modified to contain a polymer such as polyethylene glycol toreduce reticular endothelial system uptake in vivo.

Further examples of delivery vehicles comprise the use of syntheticpolymers, such as the polyamino dicarboxylic acid-co-imide derivativesdisclosed in U.S. Pat. No. 5,190,982 incorporated herein by reference orthe crosslinkable synthetic polymers such as polyphosphazines describedin U.S. Pat. No. 5,149,543 incorporated herein by reference. Anotherdelivery vehicle may comprise a liposome. In addition to the deliveryvehicles described, it is understood that any delivery vehicle designedto make hydrophobic compounds, whether they are therapeutic ordiagnostic compounds, administrable to a patient is also contemplated.

The emulsions of the present invention, whether or not they areincorporated into a delivery vehicle will generally have a size below8.0μ, and preferably below 5.0μ. It is additionally anticipated thatmicroemulsions can be prepared according to the present invention with asize below 1.0μ.

EXAMPLE 1

An emulsion useful for stabilizing the gas-forming chemical was made bymixing the following components together by rotating under vacuum.

    ______________________________________                                        Glycerol Trioleate (triolein)                                                                       1.25       g                                              1,2-dioleoyl-glycero-3-phosphocholine            15 ml                        (20 mg/ml in chloroform)                                                      cholesterol                                       0.05 g                      α-tocopherol                                  0.012 g                 ______________________________________                                    

Any remaining solvent was removed by drying under high vacuum at roomtemperature (20-25° C.). After 16 hours, 1.58 g of glycerol (1.26 g/ml)and 0.2 g perfluoropentane were added. Then, 9.6 ml of water were addedslowly while mixing at 10,000 rpm in a POLYTRON® PT3000 (Brinkman,Westbury, N.Y.) for 2 minutes at 0° C. The resultant emulsion wasfurther homogenized for 3 minutes at 30,000 rpm.

EXAMPLE 2

The ultrasonic imaging characteristics of the emulsion of Example 1 werestudied using an HP SONOS 100 Ultrasound Imaging system(Hewlett-Packard, Palo Alto, Calif.) with a 5 MHz transducer (focalzone=3.5 cm) in sector mode to detect the scattering capability of thesample solution. The compression was adjusted to obtain the greatestdynamic range possible, i.e. 60 dB. The time gain compensation controlof the ultrasound system was adjusted until the image sector beingimaged is judged visually to be optimal.

The imaging sequence was started by optimizing the instrument asdescribed on 1.0 L of water at 37° C. at 2% transmit power. A 1.0 mlsample was then injected into the water. Thereafter, every 2 minutes thetransmit power was adjusted upwards to 10, 20, 30, 40, 50, 60, 70, 80,90 and 99%. The entire sequence of images was recorded on videotape(attached to the ultrasound system) for storage and analysis.

To prepare quantitative results of this experiment, videodensitometryanalysis was performed. Selected video frames stored on the videotapewere digitized using an Apple Macintosh II computer equipped with a DataTranslation QuickCapture frame grabber board. These frames were analyzedusing CineProbe® version 1.0 (Molecular Biosystems, San Diego, Calif.)image processing software. A Region of Interest (ROI) within the beakerwas selected and the mean pixel intensity (video brightness) within theregion was determined. Each frame was then analyzed as to its meanvideodensity within the region of interest. The videodensity of a waterblank is subtracted and the resultant videodensity is expressed as VideoBrightness or Normalized Video Brightness when the initial value is setto 100 for comparison.

An ALBUNEX® (Molecular Biosystems, San Diego, Calif.) (microbubblessurrounded by a protein shell prepared as described in U.S. Pat. Nos.4,572,203; 4,718,433; 4,744,958; 4,844,882 and 4,957,656) control wasalso prepared and analyzed as described by injecting a 1.0 mL sample ofALBUNEX® (Molecular Biosystems, San Diego, Calif.) into 1.0 Liter of 37°water.

The results of this experiment are depicted in FIGS. 1A and 1B. Due tothe unchanging number of microbubbles present in the ALBUNEX® (MolecularBiosystems, San Diego, Calif.) sample, there would be expected to be alinear relationship between transmit power and video brightness. Thislinear relationship is depicted in FIG. 1A. In comparison, using theemulsion of Example 1, there would be the expectation of a bilinear orstep function between video brightness and transmit power which would bedue to some threshold energy of cavitation for microbubbles to be formedupon exposure to ultrasonic energy. Such a relationship was observed,and these results are depicted in FIG. 1B.

EXAMPLE 3

The following components were added together and homogenized in thePOLYTRON® (Brinkman, Westbury, N.Y.) at 0° C. for 3 minutes at 10,000rpm while slowly adding 10 ml ultrapure water:

    ______________________________________                                        Triolein        0.6 g                                                           Glycerol                1.57 g                                                Lecithin                0.6 g                                                 Perfluoropentane        1.5 g                                               ______________________________________                                    

These components were further homogenized for an additional 2 minutes at30,000 rpm to produce a milky white emulsion. This emulsion was filteredsuccessively through a 5μ and 1.2μ filter. The particle size wasdetermined in a Nicomp 770 (Particle Sizing Systems, Santa Barbara,Calif.) to be 95% less than 3.8μ. It was stable (no appreciable phaseseparation or particle size increase) for several days at 4° C. Whenimaged as described in Example 2, this emulsion demonstrated microbubbleformation above 40% transmit power as observed in the ultrasonic image.

EXAMPLE 4

The following components were added together and homogenized in thePOLYTRON (Brinkman, Westbury, N.Y.) at 0° C. for 3 minutes at 10,000while slowly adding 20 ml water:

    ______________________________________                                        Triolein        1.0 g                                                           Glycerol                  1.0 g                                               α-Tocopherol              0.02 g                                        PLURONIC ® F-68             0.2 g                                         Gas-forming Chemical 1.5 g of one of the                                                            following:                                              Emulsion A:  FCCl.sub.3 (Fluorotrichloromethane)                              Emulsion B:  Br.sub.2 F.sub.2 C (Dibromodifluoromethane)                      Emulsion C:  TMS (Tetramethylsilane)                                          Emulsion D:  2-Methyl butane (Isopentane)                                   ______________________________________                                    

The above emulsions were filtered through a 1.2μ filter and the particlesizes were determined as described in Example 4 to be:

    ______________________________________                                        A         95% less than 2.97 μ                                               B        95% less than 4.02 μ                                              C        95% less than 2.18 μ                                              D        95% less than 2.99 μ                                            ______________________________________                                    

EXAMPLE 5

The following components were added together and homogenized in thePOLYTRON® (Brinkman, Westbury, N.Y.) at 0° C. for 5 minutes at 10,000rpm while slowly adding 20 ml water:

    ______________________________________                                        Triolein        1.0 g                                                           Glycerol                3.0 g                                                 α-Tocopherol            0.02 g                                          Lecithin                1.0 g                                                 Perfluoropentane       1.0 g                                                ______________________________________                                    

The emulsions were further homogenized for 3 minutes at 20,000 rpm andfiltered successively through a 5μ and 1.2μ filter. The ultrasonicimaging characteristics of the emulsion was studied as described inExample 2 and exhibited microbubble formation above 40% transmit poweras observed in the ultrasonic image.

EXAMPLE 6

To further study the effects of ultrasonic energy on the production ofmicrobubbles, the emulsion of Example 5 (perfluoropentane) was imaged intwo separate experiments either continually or in 30 second intervals.For each experiment, a 1.0 ml sample of the emulsion was added to 1.0liter of water at 37° C. In the first experiment, ultrasonic imaging asdescribed in Example 2 was carried out at 99% transmit powercontinuously for 30 minutes. In the second experiment, the ultrasonicimaging was carried out for 30 second durations once every 5 minutes(intermittent imaging). Image brightness was quantified as described inExample 2 and the results are depicted in FIG. 2. These resultsdemonstrate that with continuous ultrasonic energy, due to the constantproduction of microbubbles and depletion of the bubble-formingcapability of the emulsion, image brightness was significantlydiminished at the end of 30 minutes. In comparison, with intermittentimaging which exposed the emulsions to only one tenth the energy ascompared to constant imaging (30 seconds every 5 minutes), themicrobubble-forming capability of the emulsion persisted and asubstantial amount of microbubbles continued to be produced even after30 minutes.

EXAMPLE 7

An alternative emulsion formulation comprises a viscosifier, astabilizer which is amphipathic and a gas-forming chemical formed bymixing together the following components in a final volume of 50 mLwater:

    ______________________________________                                                   Viscosifier: Stabilizer:                                           ______________________________________                                        Emulsion A   PLURONIC ® F-68                                                                          Sucrose (8.6 g)                                          (0.5 g)                                                                  Emulsion B    Sodium dodecyl-       Sucrose (8.6 g)                                sulfate (1.44 g)                                                         Emulsion C    PLURONIC ® F-68   Lactose (9.0 g)                                (0.5 g)                                                                  Emulsion D    Sodium dodecyl-        Lactose (9.0 g)                               sulfate (1.44 g)                                                       ______________________________________                                    

The solutions from above were filtered through a 0.2μ filter. A 10 mLaliquot of each of the above were mixed with 0.168 mL ofperfluoropentane in the POLYTRON® (Brinkman, Westbury, N.Y.) at 0° C.for 1 to 3 minutes at 10,000 to 20,000 rpm and then for an additional 5minutes at 20,000 rpm. Each of these four emulsions demonstratedmicrobubble formation as observed in the ultrasonic image above 40%transmit power when studied as described in Example 2.

To study the effects on these emulsions of continuous versesintermittent exposure to ultrasonic energy, a 1.0 mL sample of EmulsionC was placed in 1.0 liter of degassed water at 37° C. This solution wasultrasonically imaged either continuously or in intervals as describedin Example 6. The results are depicted in FIG. 3.

EXAMPLE 8 SYNTHESIS OF NONAFLUORO-t-BUTYLMETHANE C₄ F₉ CH₃

Starting materials (methyl iodide and cesium fluoride) were obtainedfrom Aldrich Chemical Company and perfluoroisobutylene gas was obtainedfrom Flura Corporation. Nuclear magnetic resonance spectra were obtainedusing a 200 MHz instrument tuned for determination of proton (¹ H) orfluorine (¹⁹ F) resonances.

In a flask equipped with a gas inlet, mechanical stirrer and a dry icecondenser was placed a suspension of dry cesium fluoride (42.5 g, 0.279mol) in diglyme (200 mL). Perfluoroisobutylene gas (55.5 g, 0.278 mol)was bubbled in. The gas reacted rapidly with cesium fluoride and ayellow solution resulted. The mixture was stirred for 30 minutes andthen methyl iodide (38.5 g, 0.271 mol) was added dropwise. The reactionwas slightly exothermic and the cesium iodide separated out. The mixturewas stirred for 3 hours and was allowed to stand overnight. A coldsolution (2M, sodium chloride, 500 mL) was added to the mixture withcooling (5° C.) for 30 minutes. Sodium iodide and most of the diglymesolvent dissolved in the aqueous phase which was then decanted off fromthe solid giving a crude yield of 45 g (≅40%). Distillation of thecompound sublimed at head temperature 35-39° C. and bath temperature notexceeding 50-55° C. The product was collected in a receiver cooled to-30° C. with dry ice and ethanol. The proton ¹ H NMR spectrum of itsCDCl₃ solution showed a single resonance relative to TMS; 1.65 (s, 3H,CH₃) ppm (see FIG. 4) and the ¹⁹ F spectrum, in the same solvent showedalso one single resonance at -69.99 (s, 9F) ppm relative to CDCl₃ (seeFIG. 5).

NONAFLUORO-t-BUTYLMETHANE C₄ F₉ CH₃ is shown according to either of thefollowing chemical formulas: ##STR2##

EXAMPLE 9

The following components were mixed together and homogenized in thePOLYTRON® (Brinkman, Westbury, N.Y.) at 0° C. for 3 minutes at 10,000rpm while slowly adding 10 mL ultra pure water.

    ______________________________________                                        Triolein         1.01 g                                                         Glycerol                1.05 g                                                α-Tocopherol            0.02 g                                          PLURONIC ® F-68           0.099 g                                         C.sub.4 F.sub.9 CH.sub.3                 0.780 g                            ______________________________________                                    

The resultant emulsion was filtered through a 5μ filter. The particlesize was determined as described in Example 4 to be less than 4.30microns.

The ultrasonic imaging characteristics of the emulsion were studied asdescribed in Example 2. The formation of gas bubbles was observed evenat low transmit power (<25%) settings which became brighter as thetransmit power was slowly increased to 99%.

Also for comparison a control experiment without C₄ F₉ CH₃ was conductedby mixing the following components:

    ______________________________________                                        Triolein          1.01 g                                                        Glycerol                1.05 g                                                α-Tocopherol            0.021 g                                         PLURONIC ® -F68             0.204 g                                     ______________________________________                                    

The emulsion was prepared as described above. In contrast to theprevious ultrasound imaging experiment, microbubble formation was notobserved even at 99% transmit power.

EXAMPLE 10

The following components were added together and homogenized in thePOLYTRON® (Brinkman, Westbury, N.Y.) at 0° C. for 2 minutes at 10,000rpm while slowly adding 10 ml water:

    ______________________________________                                        Triolein          1.0 g                                                         Glycerol                 1.0 g                                                α-Tocopherol            0.03 g                                          PLURONIC ® F-68              0.1 g                                        Isopentane              0.15 g                                                n-Pentane               0.85 g                                              ______________________________________                                    

The emulsion was further homogenized for 6 minutes at 30,000 rpm andfiltered through a 1.2μ filter. The ultrasonic imaging characteristicsof the emulsion was studied as described in Example 6 and a higher levelof video brightness was observed with intermittent imaging than withcontinuous imaging.

EXAMPLE 11 Emulsion-Containing Albumin Microparticle

The emulsion of the present invention can be encapsulated into adelivery vehicle comprising a multi-chamber albumin matrix as follows:

A primary emulsion is prepared by first dissolving 2.0 g human serumalbumin in 20.0 ml buffer (0.45 N Na₂ CO₃, pH 9.8) and then adding 1.0 gperfluoropentane. This mixture is emulsified in an osterizer at highspeed for 10 minutes.

A double emulsion is then prepared by adding 100 mlChloroform:Cyclohexane (1:4 v/v) with 10% (v/v) sorbitan trioleate withcontinued mixing for 10 minutes.

The albumin is cross-linked by adding, while continuing to mix, anadditional 100 ml Chloroform:Cyclohexane (1:4 v/v) containing 2.5 gterephthaloyl chloride and continuing to mix for an additional 30minutes. The reaction is quenched with 100 mL of cyclohexane containing5 g % polysorbate and 10% (v/v) ethanolamine. The microcapsules arewashed 3 times with cyclohexane:ethanol (1:1 v/v), followed by 2 washesin 5% polysorbate-95% ethanol, 2 washes in 95% ethanol and 2 washes inwater. The microparticles are then resuspended in normal saline andcomprise multi-chambered vesicles containing an inner emulsified matrixof perfluoropentane.

Although the invention has been described primarily in connection withspecial and preferred embodiments, it will be understood that it iscapable of modification without departing from the scope of theinvention. The following claims are intended to cover all variations,uses, or adaptations of the invention, following in general, theprinciples thereof and including such departures from the presentdisclosure as come within known or customary practice in the field towhich the invention pertains, or as are obvious to persons skilled inthe art.

What is claimed is:
 1. A site specific liquid-liquid emulsion for use as an ultrasonic imaging agent of a mammal comprising:a) at least one water-insoluble gas-forming chemical having a boiling point at atmospheric pressure below 37° C., wherein the chemical in said emulsion is in the liquid state at 37° C. and at atmospheric pressure in the absence of exposure to ultrasonic energy and is present in said emulsion in an amount sufficient to form microbubbles upon exposure to ultrasonic energy; and b) at least one stabilizer comprising a hydrophobic or amphipathic compound in an amount sufficient to keep the gas-forming chemical in the liquid state at 37° C. at atmospheric pressure in the absence of ultrasonic energy; wherein the emulsion forms microbubbles of gas upon application of ultrasonic energy at an energy level which is above the threshold energy of cavitation for microbubbles to be formed.
 2. A site specific liquid-liquid emulsion for use as an ultrasonic imaging agent of a mammal comprising:a) at least one water-insoluble gas-forming chemical having a boiling point at atmospheric pressure below 37° C., wherein the chemical in said emulsion is in the liquid state at 37° C. and at atmospheric pressure in the absence of exposure to ultrasonic energy and is present in said emulsion in an amount sufficient to form microbubbles upon exposure to ultrasonic energy; and b) at least one stabilizer comprising a hydrophobic or amphipathic compound in an amount sufficient to keep the gas-forming chemical in the liquid state at 37° C. at atmospheric pressure in the absence of ultrasonic energy; wherein the emulsion forms microbubbles of gas upon application of ultrasonic energy in vitro to a mixture of said emulsion and water at a ratio of 1:1000 (v/v) emulsion:water at 37° C.
 3. A method of forming microbubbles in an animal comprising:a) injecting the animal with a liquid-liquid emulsion capable of forming microbubbles upon application of ultrasound energy at 37° C.; and b) applying ultrasound energy in an amount sufficient to cause formation of microbubbles; wherein the emulsion comprises at least one water-insoluble gas-forming chemical having a boiling point at atmospheric pressure below 37° C., wherein the chemical in said emulsion is in the liquid state at 37° C. and at atmospheric pressure in the absence of exposure to ultrasonic energy and is present in said emulsion in an amount sufficient to form microbubbles upon exposure to ultrasonic energy; and at least one stabilizer comprising a hydrophobic or amphipathic compound in an amount sufficient to keep the gas-forming chemical in the liquid state at 37° C. at atmospheric pressure in the absence of ultrasonic energy. 